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The Limulus Amebocyte Lysate (LAL) test

What is the LAL test, what are its strengths and weaknesses and how the LAL test compares to other endotoxin and pyrogen tests available on the market.


What is the LAL test?

The LAL test is a bacterial endotoxin test (BET) employed by medicinal product manufacturers worldwide. Specifically, the LAL test is a means of detecting and in some cases quantifying the presence of endotoxins, also known as lipopolysaccharides (LPS), deriving from gram negative bacteria. The LAL test is employed as a batch release assay for parenteral drugs and medical devices before their distribution into the market. 

Background of the LAL test

Frederick Bang and Jack Levin observed in the 1950s that amebocytes of the horseshoe crab, Limulus polyphemus, clot when exposed to endotoxins. Within 30 years, the LAL test was introduced across pharmacopoeias as an endotoxin detection assay. Though initially hoped to replace the rabbit pyrogen test (RPT), the LAL tests failure to detect non-endotoxins pyrogens meant it could not gain status as a stand-alone pyrogen test. 


How does the the LAL test principle work?

A drug sample is incubated within the LAL test reagent. If the drug sample is contaminated with endotoxins, a process initiates within the horseshoe crab amebocytes involving the chained activation of a cascade of enzymes, which culminates in clotting. In the case of classical LAL, the clotting that is observed signals endotoxin contamination.

Does the Ph. Eur. approve the LAL test?

The European Pharmacopoeia approves the LAL test as a compendial assay for Endotoxin detection (2.6.32). Seeing as the LAL test does not constitute a pyrogen test however, the European Pharmacopoeia (5.1.10) mandates the necessity for a test-based risk assessment for non-endotoxin pyrogens to also occur at release or during development of the production process wherever an Endotoxin test like the LAL test is employed at release.


Variations of the LAL test include —

There are predominantly three variations of the LAL test technique: the gel-clot, turbidimetric and chromogenic methods. Beyond these, the latter two have further sub-variants; kinetic and endpoint tests.


The gel-clot LAL test assay

The gel-clot LAL test method requires no detection equipment or software, simply the human eye to determine whether gel clotting has taken place after drug incubation. This purely qualitative LAL test method is simple, economical but low on sensitivity (LoD of 0.015 - 0.5 EU/ml); adequate for just swift and informal testing during the initial stages of a product's development. 


The turbidimetric LAL test assay

The turbidimetric LAL test method employs a photometric instrument to kinetically read the cloudiness (turbidity) of the clotted test solution. This LAL variant is more sensitive than the gel-clot (LoD of 0.001 EU/ml) method and delivers quantifiable results, making it suitable for endotoxin release testing of simple pharmaceutical products.


The chromogenic LAL test assay

The chromogenic LAL test method uses a tube or microplate reader to visualize and measure the degree of color change that is induced during the clotting process. The most sensitive (LoD of 0.0002 EU/ml) but expensive LAL variant, this method delivers quantifiable results, making it best suited as an endotoxin detecting release assay for today's more complex drugs and medical devices.



Further LAL test sub-variants

There are two different methods for the turbidimetric and chromogenic LAL tests: kinetic and endpoint. The kinetic method measures endotoxins at different intervals during the assay's reaction while the endpoint method takes measurement only after the reaction is finished. The endpoint method's single measurement means it must be taken at exactly the right time for it to succeed otherwise the test must be repeated.

Specifics to note when considering whether to employ the LAL test

Human non-specificity

Unlike other assays, the LAL test does not mimic the human reaction to endotoxin contamination as it is based on horseshoe crab, rather than human, blood.

In-vitro but still animal harming

Biomedical companies are said to harvest only 30% of the blood of a caught horseshoe crab when producing their LAL tests and then return it to the ocean. However, a growing body of evidence shows that 5 - 20% of crabs die during the extraction process while those that return to the ocean experience significant difficulty to reproduce.

Omission of NEP detection

While the LAL test is efficacious in detecting endotoxin pyrogens deriving from gram negative bacteria, it fails to detect non-endotoxin pyrogens that may contaminate a parenteral which derive from gram-positive bacteria, plastics, molds and a wide host of other sources.

Susceptibility to LER effects

Although our understanding of the exact underpinnings of the LER effect is still incomplete, what we do know is that the LAL's enzymatic reaction make it particularly susceptible to such masking effects when compared to other assays based on biological reactions (e.g. MAT).

The LAL Test vs. other test methods

The LAL test vs. RPT

Even though RPT has a much lower sensitivity than LAL, RPT does test (like MAT) for both endotoxins and non-endotoxin pyrogens (NEPs) as a qualitative test. The Limulus Lysate Test can only test for endotoxins, but is very capable of doing so. That’s why in 1977 the USFDA allowed LAL to be a replacement of RPT for detecting endotoxins in pharmaceutical and medical devices. All international pharmacopeias recommend LAL as the method to test for endotoxins. Even so, it is still a requirement in the US to test a new drug for NEPs at least once. Many pharmacopeias also recommend to, wherever possible, replace tests that make use of animals with tests that don’t, and both RPT and LAL make use of animals for their tests. LAL however, takes the blood from the horseshoe crab and uses it outside of the living body, whereas with RPT the drugs that need to be tested are injected into the rabbits

The LAL test vs. rFC

An alternative BET method, based on LAL, has been recently developed. This method uses a recombinant (genetically engineered) form of factor C, the first enzyme in the horseshoe crab clotting cascade. But with rFC this cascade of multiple enzymatic steps doesn’t take place; instead, it’s a single enzymatic step. It is supposed to be as accurate, sensitive and specific as LAL. Because factor C is genetically engineered, it’s an animal-free method. Nevertheless, it is still based on the process that takes place inside of the horseshoe crab, and unlike MAT, not based on the human reaction to endotoxins. Besides that, rFC like LAL, can not detect non-endotoxins.

The LAL test vs. MAT

The biggest differences between LAL and MAT concern the use of animals, and whether they are able to test for non-endotoxins or not. While LAL takes blood from the horseshoe crab to test for endotoxins, MAT uses human donor blood to mimic the human reaction to pyrogens; endotoxins and non-endotoxin pyrogens alike. As a quantitative test, MAT reveals the amount of pyrogens present, as well as the effect it has on human beings. Therefore, MAT is being transitioned in by many pharmacopoeias. Additionally, MAT has been approved by the EP as an ample pyrogen test, whereas with the LAL test you’re still required to test for non-endotoxins at least once (using RPT or MAT). 


0.005 - 50.0 EU/ml

Inadequate for pyrogen testing




Assay analysis


Amebocyte Lysate Test (LAL)


Pyrogen Test (RPT)

Monocyte Activation Test (MAT)

Assay type

Sensitivity (Limit of Detection)



Ph. Eur. perspective



0.05 EU/ml

0.005 EU/ml

Quantitative Qualitative




Inadequate for pyrogen testing





0.005 EU/ml

Inadequate for pyrogen testing




Detectable pyrogenic contaminations

Endotoxins (from
Gram negative

Yeasts & Molds




Possible applications




Medical devices

Cell therapeutics






Recombinant factor C (rFC)

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